The present invention relates to a method of releasing solid matrix affinity adsorbed particulates by enzymatically degrading the solid matrix to which the particulates are adsorbed. More particularly, the present invention relates to a method of releasing particulates, such as viruses, cells or fragments thereof, which are affinity adsorbed on a polysaccharide solid matrix, by enzymatically degrading the polysaccharide solid matrix to which the particulates are adsorbed with a polysaccharidase.
The need for efficient affinity adsorption and release methods:
There is a tremendous effort being made to develop rapid cell testing and separation methods to meet the needs of the food, medical, environmental and veterinary industries.
The food industry, for example, needs rapid microbial testing to approve or reject raw materials and determine whether or not to release a held batch of product. Furthermore, with the increasing implementation of hazard analysis and critical control point programs by the food industry, the demand for rapid microbial testing has been steadily on the rise (Rules and Regulations, Department of Agriculture Food Safety and Inspection Service, "Pathogen Reduction; Hazard Analysis and Critical Control Point (HACCP) Systems" (1996) 61 Federal Register 38806).
Rapid microbiological methods such as nucleic acid probe hybridization and immunological assays have advanced dramatically, shortening the time required for the detection of pathogens in meats and other foods.
However, these methods require concentrations of target microorganism of 10.sup.4 to 10.sup.6 cells per ml or more (Blackburn et al., 1994, Lett. Appl. Microbiol. 19:32-36; Swaminathan et al., 1994, Ann. Rev. Microbiol. 48:401-426; and Tian et al., 1996, J. Food Prot. 59:1158-1163). Foods which are contaminated by bacterial pathogens usually have low numbers of bacteria so that an enrichment step is required prior to the application of a rapid detection assay or even a selective culture method.
PCR-based assays have the potential to overcome the need for long selective enrichment steps due to their ability to detect and identify pathogens in the presence of large numbers of background flora (K. Venkateswaran et al., 1997, Appl. Environ. Microbiol. 63:4127-4131).
Nevertheless, when target pathogen concentrations are low, i.e., less than 10.sup.3 CFU/g, current PCR procedures require 6 to 18 hours of enrichment prior to PCR amplification in order to detect such target pathogens. This enrichment step not only brings the target pathogen, which may be present at levels of less than 1 cell per ml or gram of food (Swaminathan et al., 1994, Ann. Rev. Microbiol. 48:401426), to PCR detectable levels but also allows for samples to be diluted or filtered to reduce or partially remove PCR inhibitory food components while bringing the target pathogen to a concentration which is detectable (K. Venkateswaran et al., 1997, Appl. Environ. Microbiol. 63:4127-31).
In culture methods, selective reagents are necessary in order to inhibit the growth of competing microorganisms but they also inhibit target bacteria ultimately increasing the time it requires to achieve detectable levels of the target pathogen. Similarly, many foods contain compounds which are inhibitory to the target bacteria and in many cases the target bacterial cells do not revive or revive very slowly. Furthermore, selection media inhibit the "resuscitation" (reviving the viability) of damaged bacteria present, e.g., in meat samples due to freeze thaw cycles, inhibitory food components, or desiccation during processing.
The possibility of selectively concentrating and thereafter recovering and counting or analyzing target bacteria from a food sample, other than by culture methods, while removing background flora and inhibitory compounds would have a tremendous impact on rapid food testing saving time and increasing sensitivities of existing detection techniques.
Zero tolerance standards require the detection of these damaged pathogens even when they are present at minute levels. On the other hand, selective concentration of target bacteria would enable rapid enrichment to be carried out without the addition of selective reagents which are necessary in order to inhibit the growth of background flora but which also inhibit target bacteria and ultimately increase the time it takes to achieve detectable levels of target pathogen. Removal of competing microorganisms and inhibitory compounds found in food samples to affect resuscitation of target pathogens is best accomplished by selective concentration and subsequent wash steps.
In summary, efficient methods for selective affinity concentration and release of a particular pathogen(s) from foods would not only shorten the overall time required for detection of these pathogens, but would allow for more sensitive detection due to more efficient resuscitation of damaged bacteria.
Furthermore, the ability to collect larger more representative samples while selectively concentrating and recovering a specific pathogen from these samples would greatly increase the probability of detecting a pathogen in foods when they are present at extremely low concentrations.
Methods of efficient affinity adsorption and concentration of a microorganism or microorganisms are disclosed in U.S. patent application Ser. No. 09/175,040, filed Oct. 19, 1998, which is incorporated by reference as if fully set forth herein.
This application teaches methods for concentrating a particular microorganism or microorganisms of interest in a sample. The disclosed methods are generally effected by contacting a sample with a cellulosic or chitin matrix to which is bound a cellulose binding protein ("CBP")-receptor (i.e., a first member of a binding pair) or cellulose binding domain ("CBD")-receptor conjugate specific for said microorganism(s).
The methods also include a washing step to remove unbound material of the sample from the matrix. The methods also include an optional step for enriching the concentrated microorganism(s) in situ by addition of a culture medium to the matrix or by enriching the concentrated microorganism(s) in vitro by transferring the microorganism(s) from the matrix to a culture medium. The method also includes an optional step of performing an assay to detect any microorganisms that bind to the CBP- or CBD-receptor conjugate bound to the matrix.
The disclosed methods have utility in concentrating microorganisms in samples, particularly dilute samples, in order to detect the microorganisms by any means known in the art. Such methods of concentration provide improved means of concentrating microorganisms in food, environmental, or biological, such as medical or veterinary, samples.
The disclosed methods have a number of advantages over previously described concentration methods. For example, the use of cellulosic fabric as a matrix allows for larger volumes of liquids (up to 10 liters) to be passed with relatively high flow rates as compared to, for example, the DYNAL.RTM. DYNALBEADSO.RTM. procedure [Dynal-product Cat. No. 710-03]. The low non-specific binding of the cellulose achieves very low background levels. In certain preferred embodiments, the disclosed methods are able to capture microorganisms present at very low concentrations by use of high surface area cellulosic or chitin matrix, such as, but not limited to gauze. The physical properties of the cellulosic or chitin matrix enable its performance under conditions that Immuno Magnetic Separation (IMS) do not perform effectively, i.e., in the presence of food samples containing milk and food samples containing bacteria at concentrations lower than 10.sup.3 CFU/ml.
However, no efficient method of releasing the captured microorganism is disclosed in U.S. patent application Ser. No. 09/175,040.
There is also a need for efficient adsorption and release of eukaryotic cells. Of particular interest are bone marrow and hematopoietic derived cells. Particular cell types derived from these sources, such as, but not limited to, hematopoietic stem cells, stromal stem cells, lymphocytes, etc., are used in a variety of therapeutic allogenic and autologous procedures as well as diagnostic procedures. Examples include, but are not limited to, adoptive immunotherapy for treatment of cancers, cartilage damage repair for treatment of damaged joints, gene therapy for treatment of genetic disorders and cancer, analysis of maternal blood derived fetal cells for detection of genetic diseases, removal of cells causing graft vs. host disease in cases of bone marrow transplantation. In most cases, however, the cell types required for such procedures are scarce and need to be separated from a mixed cell population. Affinity adsorption is in many cases employed to separate the required cells from the mixed cell population.
An efficient method of affinity adsorption and concentration of eukaryotic cells is disclosed in PCT/CA97/00033 (WO 97/26358), which is incorporated by reference as if fully set forth herein. This application teaches methods and compositions for isolating growth-factor dependent cells through the use of immobilized growth factors. The compositions include a matrix binding polypeptide and a growth factor conjugate which is used as an affinity complex to adsorb the growth factor dependent cells to the matrix.
Prior art methods of affinity adsorption and release of cells:
The technology for capturing specific cells on affinity matrices is well developed. To this end, see, for example, Wigzel, et al. (1969), J. Exp. Med., 129:23; Schlossman, et al. (1973), J. Immunol., 110:313; Mage, et al. (1977), J. Immunol. Meth., 15:47; Wysocki, et al. (1978), Proc. Nat. Acad. Sci., 75:2844; Schrempf-Decker, et al. (1980), J. Immunol. Meth., 32:285; Muller-Sieburg, et al. (1986), Cell, 44:653, which are incorporated herein by reference.
Various methods have been proposed to effect the release of cells or other target substances from a solid matrix once they have been exclusively adsorbed thereto. U.S. Pat. No. 3,970,518 to Giaever discloses the use of antibody-coated magnetic microspheres to separate cells, and uses a chemical cleaving agent such as formic or sulfuric acids to release the separated cells. U.S. Pat. No. 4,988,621 to Hayman, et al. discloses the use of a short peptide which interferes with the binding of cells to fibronectin, allowing the detachment of cells from a solid matrix. EP 463508 A to Mori discloses the use of a temperature-responsive adhesive to immobilize a cell for microinjection, lowering the temperature to release the immobilized cell. WO 91/16452 to Berenson describes a technique involving agitation of an avidin column to remove cells captured thereon. The use of ionic strength manipulation to reversibly immobilize antibodies bound to magnetic beads has also been reported. See, for example, Scouten, Anal. Biochem. 205:313-18 (1992). U.S. Pat. No. 5,081,030 to Civin discloses the use of chymopapain to digest the cell surface antigen My10, releasing stem cells from magnetic particles used to isolate the stem cells from a cell suspension. WO 94/20858 to Berge et al. describes the separation of target substances by means of a relatively large magnetic particle linked via a hydroxyboryl/cis-diol bond to an antibody, which bond is cleaved after separation of the target substance. The commercially available DETACHaBEAD from Dynal (Oslo, Norway) comprises an anti-mouse FAb with higher affinity for the binding site of a monoclonal antibody than the monoclonal has for its corresponding antigen. Therefore, the anti-FAb antibody can displace the original MAb from a target cell. See Geretti, et al. J. Immunol. Meth. 161:129-31 (1993); and Rasmussen, et al., J. Immunol. Meth. 146:195-202 (1992). WO 94/02016 to Kesler describes the use of an excess of soluble hapten to disrupt a hapten-antihapten complex, thereby releasing a cell from its solid matrix. See also Clark et al., J. Immunol. Meth., 51:167-70 (1982). Competitive affinity elution is disclosed by Grandics, et al. (U.S. Pat. No. 5,773,224).
Some other proposed methods include reduction of disulfides or cleavage of specific linkages by enzymatic or chemical agents inserted somewhere between the capture solid surface and the target cells. Such techniques may include protease digestion of marker antibodies or coupling peptides, cellular oligosaccharides cleavable with glycolytic enzymes, or chemical bonds broken under mild conditions that will preserve biological activity, such as oxidizing, reducing, basic, or acidic conditions. Because of the extreme complexity of cell surfaces and the desire to maintain high cell viability and functional integrity during selection, it is very difficult to find an enzymatic or chemical release method which has no effect on the cell structure and/or function. Mechanical agitation/elution can be damaging to the cell membrane and reduce cell viability.
All of the above methods suffer one or more disadvantages, which may include, low release/elution yields, non-stoichiometric release/elution, low recovery following elution and cell damage. However, all of the above methods share a common limitation--they lack universality.
The following paragraphs provide some more insight into the limitations associated with various prior art approaches of releasing particulates adsorbed to solid matrices.
Thus, a variety of matrices have been employed for capturing specific cells for the isolation thereof from mixed cell populations. In all of these cases the release of the cells involve perturbation of the cell surface or destruction of the immobilizing linker. Each of these treatments can be detrimental to the viability of the recovered cells.
Current methods for releasing cells from surfaces to which they are adsorbed include, for example, chemical treatment, heat treatment, enzymatic treatment, competitive release and release via receptor internalization. Each of these methods suffer limitations as further detailed hereinunder.
Chemical and heat treatments:
Examples for methods which are used to release cells from antibodies bound to a solid matrix include treatment with chaotropic agents, such as, but not limited to, 4.5 M MgCl.sub.2 pH 7.5, or 2.5 M NaI pH 7.5 (U.S. Pat. No. 5,415,997), polarity reducing agents, such as ethylene glycol in solutions of up to 50% (U.S. Pat. No. 5,415,997), and agents that lead to changes in pH, such as glycine/HCl pH 2.5, aqueous NH.sub.3 pH 11, and 0.5% KOH pH 12.5 (U.S. Pat. No. 5,415,997), or in ionic strength, such as in the case of oligo dA tagged antibodies which are bound to Dynabeads Oligo-dT. In all of the above cases the viability of the target cells is compromised.
Other methods exist which can be used to release cells which have been captured by different means on a wide variety of solid matrices. High temperatures and other harsh treatments such as lytic reagents (NaOH) and denaturing reagents (5 M urea, 6 M guanidine HCl) can also lead to the release of cells from the matrices to which they are bound, however these cells are not viable. These approaches are suitable if the goal is to perform tests such as DNA analysis or immunoassays for specific antigen of the organisms which are released from the matrix, which can be performed using a non-viable cellular material. However these methods are not suitable for the recovery of viable cells and in some cases the antigen which is the target of an immunoassay may be destroyed by the releasing agent.
It should be noted that even in cases where viable cells are not directly required to perform a test, it may still be desirable to amplify the recovered cells before running the test. This is especially true when the number of cells captured on the matrix is less than the amount required to generate a sufficient signal to give a statistically significant positive result in the test selected.
Elution methods which have a lethal effect on the viability of the targeted cells generates a need for capturing extremely high numbers of target cells in order to recover just a few after elution. Often this demand is answered by prolonged enrichment steps prior to the capture of the target cells. However, these enrichment steps often cause exponential growth of unwanted microorganisms as well, which form a burden to the capture system and its ability to produce isolated target cells following the elution step.
Enzymatic treatment:
In the case of linker-based immobilization through a cell specific antibody or ligand, it is often possible to digest the linker molecule itself. This can be done with general proteases such as proteinase K or pronase, however these enzymes tend to digest proteins nonspecifically, and typically damage cell surface proteins which leads to loss of viability and/or functional activity of the cell surface protein. Even more specific proteases targeted at specific portions of an antibody or ligand often have detrimental affect on cell viability. Furthermore, these specific enzymes are not necessarily very efficient at mediating the quantitative release of cells from the matrix. In general, the efficiency of a particular protease for the release of a cell which has been captured by an antibody or other ligand must be determined empirically. Not every protease performs with every antibody, ligand and/or coupling method. In some cases, the protease concentration required to effect cell release is so high that cell is viability is compromised.
Competitive release:
In some cases, reagents are available which can compete off the bound cells. These may be high concentrations and/or slightly altered soluble forms of the immobilized antibody or ligand. Unfortunately, this approach is often unsuccessful in practice. This may be due, in part, to the fact that the density of the cell surface molecules leads to very high avidity multivalent interactions with the immobilized antibodies or ligands which often require drastic elution conditions which prevent viable cell recovery (Bonnafous J C, et al., J Immunol Methods, 1983 58 (1-2):93-107). Furthermore, it may be necessary in some cases to produce a competitive reagent specifically for each ligand-target cell pair, rendering this option cost-ineffective and/or impractical. An example of competitive release is shown with the use of methoxyethoxymethyl (MEM) bonded-phase Cellulofine columns to selectively separate human peripheral platelets, granulocytes and lymphocytes. Selective elution was achieved by using different mobile phases containing various saccharides (Shibusawa et al., J. Chromatogr. B. Biomed. Appl. 1995, 666(2):233-239).
Receptor internalization: Release, can in some rare cases, be obtained by simply incubating cells which have been captured over night in cell culture. B cells can be released from Dynabeads after capture by overnight incubation in cell culture which causes transient down regulation of the target surface antigen by the target cell. Again, this method is limited to specific cell types which can and are induced to perform internalization under capture conditions.
There is thus a widely recognized need for, and it would be highly advantageous to have a method of releasing solid matrix affinity adsorbed particulates by enzymatically degrading the solid matrix to which the particulates are adsorbed, because such a method is both universal and it avoids the above limitations associated with prior art release methods.